title: Functional role and analysis of cysteine residues of the salt tolerance protein Sod2.
abstract: Sod2 is the major salt tolerance plasma membrane protein of Schizosaccharomyces pombe. It functions to remove excess intracellular sodium (or lithium) in exchange for protons. We investigated the role of cysteine residues and created a cysteine-free Sod2 protein. Each cysteine residue of the ten present was individually mutated to serine and the different proteins expressed and characterized in S. pombe. Western blotting revealed that all the individual mutant proteins were expressed. We examined the ability of the mutant proteins to confer salt tolerance to S. pombe with the endogenous Sod2 protein deleted. Only proteins with C26S and C374S mutations were partially reduced in their ability to confer salt tolerance. Additionally, they showed a change in conformation in comparison to the wild-type protein, indicated by differential sensitivity to trypsin. Deletion of all the cysteine residues of Sod2 resulted in a functional protein that was expressed in S. pombe at levels similar to the wild type and also conferred salt tolerance. The conformation of the cysteine-free Sod2 protein was not altered relative to the wild-type protein. We examined the accessibility of amino acids of the cysteineless protein present on putative extracellular loop 2. A cysteine placed at position Ala119 was accessible to externally applied [2-(trimethylammonium)ethyl] methane thiosulfonate bromide. The results demonstrate that cysteines in the Sod2 protein can be changed to serine residues resulting in an expressed, functional protein. The utility of the cysteine-free Sod2 protein for determination of topology and amino acid accessibility is demonstrated.
authors.affiliation: Department of Biochemistry, University of Alberta, 347 Medical Science Building, Edmonton, AB, T6G 2H7, Canada.
authors.forename: Asad
authors.initials: A
authors.lastname: Ullah
authors.forename: Rabab Abou
authors.initials: RA
authors.lastname: El-Magd
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000222:Adaptation, Physiological; D001483:Base Sequence; D015153:Blotting, Western; D003545:Cysteine / Q000502:physiology*; D017931:DNA Primers; D016297:Mutagenesis, Site-Directed; D012568:Schizosaccharomyces / Q000254:growth & development / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012965:Sodium Chloride; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D017931:DNA Primers; D029702:Schizosaccharomyces pombe Proteins; C501077:Sod2 protein, S pombe; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride; D014357:Trypsin; D003545:Cysteine